Effect of HIF-1alpha suppression on ING4 and the angiogenic-related molecules in hypoxic condition. JJN3 was transfected by electroporation with 1 nmol smart pool double-stranded RNA oligonucleotides (siRNA) against HIF-1alpha or with a nonspecific control siRNA (Cy). After 24 hours, cells were incubated in the presence or absence of hypoxic condition (1% O2, 5% CO2 atmosphere or CoCl2 treatment) for 12 hours. HIF-1alpha mRNA expression was evaluated by RT-PCR, whereas HIF-1alpha protein level and activity were detected by Western blot and ELISA, respectively, as described in “Western blot analysis” and “ELISAs.” Graphs represent the means plus or minus SD of 2 independent experiments measured in triplicate (OD = optical density). Nuclear extracts of COS-7 treated with CoCl2 in the presence or absence of wild-type (wt) or mutated (mt) competitor oligonucleotides were tested as control (Con) (A). Aliquots of conditioned media of JJN3 transfected cells were tested by ELISA to detect IL-8 and OPN levels. Graphs represent the means plus or minus SD of IL-8 or OPN levels in 2 independent experiments measured in triplicate (B). JJN3 was transfected by electroporation with 1 nmol smart pool double-stranded RNA oligonucleotides (siRNA) against HIF-1alpha or ING4 or HIF-1alpha plus ING4 or with a nonspecific control siRNA (Cy) and incubated after 24 hours in the presence or absence of hypoxic condition. Thereafter ING4, IL-8, OPN, HIF-1alpha, and NIP-3 mRNA expression was quantified by realtime PCR. Data are expressed as mean −deltaCt's plus or minus SD as described in “Real-time quantitative PCR” (C).
